Cyclic analogs of atrial natriuretic peptides

ABSTRACT

Compounds and compositions comprising cyclic synthetic analogs of Atrial Natriuretic Peptides are provided, together with methods for their production and use as natriuretics, diuretics and/or vasodilators, or as intermediates for or modulators of such useful compounds or of native Atrial Natriuretic Peptides.

This is a continuation-in-part of U.S. patent application Ser. No.07/138,893, filed 24 Dec. 1987, abandoned, which is acontinuation-in-part of the parent of U.S. patent application Ser. No.07/168,661, filed 16 Mar. 1988, now U.S. Pat. No. 4,804,650, which is acontinuation of U.S. patent application Ser. No. 06/921,360, filed 28Oct. 1986, abandoned, which is a continuation-in-part of U.S. patentapplication Ser. No. 06/904,091, filed 4 Sept. 1986, abandoned, which isa continuation-in-part of U.S. patent application Ser. No. 06/868,312,filed 28 May 1986, now U.S. Pat. No. 4,757,048, which is acontinuation-in-part of U.S. patent application Ser. No. 06/795,220,filed 5 Nov. 1985, abandoned.

TECHNICAL FIELD

The present invention relates generally to synthetic analogs of atrialpeptides and more particularly to synthetic peptide compounds which finduse as diuretics, natriuretics and/or vasodilators, or as intermediatesfor or modulators of such useful compounds, together with methods fortheir production and use.

BACKGROUND ART

Most multi-cellular organisms are organized into tissues and organswhich perform specialized functions. Thus, a system has evolved totransport and circulate materials between them. In higher animals,including mammals, this circulatory system is closed, in order toimprove the efficiency of transport. The flow of blood fluid throughthis closed cardiovascular system requires that the fluid be maintainedunder pressure and the regulation of the systemic arterial bloodpressure requires a complex interaction of numerous factors including,e.g., fluid volume and vascular elasticity and caliber.

The maintenance of normal extracellular fluid volume depends primarilyon the excretion of sodium (natriuresis) and water (diuresis) by thekidneys. This is determined by (1) the rate at which plasma is filteredat the glomerulus (glomerular filtration rate, or GFR) and (2) thedegree to which sodium is actively reabsorbed along the renal tubule(with water following passively). The latter process is in partregulated by the adrenal steroid hormone aldosterone. It has been longbelieved that, in addition to GFR and aldosterone, there must be a"third factor" which also regulates sodium reabsorption. It is nowapparent that many of the phenomena which required the postulation of a"third factor" can be explained by the effects of physical forces (e.g.,blood pressure, red blood cell concentration and plasma viscosity) onsodium reabsorption. Nonetheless, the search continues for a"natriuretic hormone" which might modulate tubular reabsorption.

A natriuretic effect has been demonstrated by crude extracts of ratatrial tissue but not ventricular tissue. De Bold, A. J., et al., LifeSciences, 28: 89-94 (1981), Garcia, R., Experientia, 38: 1071-73 (1982),Currie, M. G., et al., Science 221: 71-73 (1983). Various peptides withdiuretic and natriuretic properties have been isolated from atrialtissue and sequenced. Flynn, T. G., et al., Biochem. Biophys. Res.Commun. 117: 859-865f (1983), Currie, M. G., et al., Science 223: 67-69(1984), Kangawa, K., et al., Biochem. Biophys. Res. Commun. 118: 131-139(1984).

More recently, various seemingly related peptides have been isolated,sequenced and shown to have natriuretic, diuretic and vasorelaxantactivity in varying degrees. U.S. Pat. No. 4,496,544: U.S. Pat. No.4,508,712; Kangawa, K., et al., Biochem. Biophys. Res. Commun. 121(2):585-591 (1984); Kangawa, K., et al., Biochem. Biophys. Res. Commun.119(3): 933-940; Garcia, R., et al., Biochem. Biophys. Res. Commun.126(1): 178-184 (1985); Katsube, N., et al., Biochem. Biophys. Res.Commun. 128(1): 325-330 (1985).

The existence of these atrial natriuretic factors strengthens thelong-held suspicion that the heart, aside from its obvious influence onrenal perfusion, plays an important role in regulating renal sodium andwater excretion.

A number of clinically important disease states are characterized byabnormal fluid volume retention. Congestive heart failure, cirrhosis ofthe liver and the nephrotic syndrome each lead to excessive fluidaccumulation on the venous side of the circulation, the presumed commonmechanism being under-perfusion of the kidneys leading to a fall in GFR.In addition, the reduced renal perfusion stimulates excessive secretionof renin, a proteolytic enzyme whose action in the circulation leads tothe formation of angiotensin. Angiotensin is a powerful constrictor ofarterioles (which helps to maintain arterial pressure) and alsostimulates release of the sodium-retaining hormone aldosterone by theadrenal gland (which further worsens fluid retention). These mechanismsdo not, however, fully account for the fluid retention of the so-called"edematous states," and additional factors are likely to be involved.

An increase in extracellular fluid volume is also thought to contributeto the development of hypertension in many instances. Hypertension, orchronically elevated blood pressure, is one of the major causes ofillness and death worldwide. It is estimated that more than 20 millionAmericans suffer from this disease whose complications include heartfailure, heart attack, stroke and kidney failure. The major observedhemodynamic abnormality in chronic hypertension is increased resistanceto the flow of blood through the arterioles. The mechanisms which leadto this increased "peripheral resistance" are, however, incompletelyunderstood. In some cases inappropriate activity of therenin-angiotensin system or sympathetic nervous system may lead toexcessive constriction of the arterioles; by "inappropriate" it is meantthat the unknown signal(s) leading to this activity are not based upon aphysiological need of the organism, and thus lead to elevated bloodpressure. In a substantial fraction of hypertensives, however,inappropriate sodium and volume retention by the kidney is felt toeither initiate or contribute to the elevated blood pressure. Theresponsible defect in kidney function and the mechanism whereby fluidretention leads to increased peripheral resistance are both unknown. Itis possible that a relative deficiency of a natriuretic hormone could beresponsible for these observations, particularly if the same substancealso normally exerted a relaxant effect on arterioles.

Diuretic therapy is currently a mainstay in the treatment ofhypertension, renal failure and the various edematous states (heartfailure, etc.). Currently available pharmacological preparations have,however, several important limitations and undesirable effects. Whiletheir use may be directed at a specific abnormality (i.e., volumeexpansion), their multiple actions are undoubtedly not physiological,leading for instance to potassium depletion, increased retention of uricacid and abnormal glucose and lipid metabolism. In addition, all knowndiuretics profoundly stimulate the renin-angiotensin-aldosterone system,which counteracts their volume-depleting and blood pressure-loweringeffects and leads to other unwanted effects. It would be desirable toprovide a pharmacologically effective compound which can regulate bloodpressure by providing a complete but controlled range of physiologicalresponses.

However, the isolation of such compounds from atrial tissue is typicallya cumbersome process and requires substantial substrate tissue toproduce minute quantities of the compounds.

Furthermore, it is considered desirable to provide modifications to thenative structures reported for these atrial natriuretic factors in orderto isolate the regions of the peptides responsible for the distinctbiological activities or regions important in the metabolism andclearance of the peptide. Having determined the appropriate units ofactivity, structural analogs can be created which preserve, e.g.,natriuretic or diuretic activity. Furthermore, shortened peptidesequences will provide active synthetic analogs which can be takenorally or delivered intranasally to provide the therapeutic benefits ofthe native compositions.

Shortened and modified peptide sequences will also desirable beformulated to enhance their direct or indirect biological activity,resistance to degradation, biological half-life and to enable thechemosynthetic production of these compounds in a cost-effective mannerfor clinical use.

DISCLOSURE OF THE INVENTION

It has now been found that a class of synthetic analogs of native AtrialNatriuretic Peptides (ANPs) which have been prepared in accordance withthe present invention is capable of exhibiting or modulating thenatriuretic, diuretic and/or vasorelaxant activity of the endogenous ornative peptides in mammals in vivo.

Most of the synthetic analog compounds of the present invention retain acore pentapeptide sequence of amino acid residues which correspond in adefined way to the sequence AA₈ -AA₁₂ of native ANPs, using theidentification system from Atlas, S., et al., Nature 309: 717-719 (1984)wherein the amino-terminal arginine residue is at position 1. In theknown native ANPs, this core sequence is RIDRI in rat and RMDRI inhuman. Certain defined permutations of this sequence, including somewherein AA₁₂ is not present, retain activity in vivo and demonstratethat the core peptide structure is a significant factor in the peptide'sbiological activity. However, as explained hereinbelow, many of thesecompounds are not active in in vivo model systems for assay of diureticor natriuretic activities. It is likely that these analogs empower thefunction of endogenous ANPs by blocking clearance receptor(s) for thesepeptides.

The native ANPs are cyclic disulfides. The compounds of the inventionare cyclic, but need not be disulfides, as set forth hereinbelow.

The present invention is, therefore, in one aspect directed to cyclicanalog peptide compounds having natriuretic, diuretic and/orvasorelaxant activity in mammals which have the formula:

    X.sub.1 -AA.sub.y -X.sub.2 -AA.sub.8 -AA.sub.9 -AA.sub.10 -AA.sub.11 AA.sub.12 -X.sub.3 -AA.sub.z -X.sub.4                     ( 1)

wherein:

each of AA₈ and AA₁₁ is, independently, a basic/noncyclic, but can bealso a neutral/nonpolar/small or neutral/polar/large/nonaromatic aminoacid residue;

AA₉ is a neutral/nonpolar/large/nonaromatic amino acid residue in the Dor L configuration;

AA₁₀ is an acidic amino acid residue; and

AA₁₂ is a neutral/nonpolar/large/nonaromatic amino acid residue, in theD or L configuration or is a covalent bond;

AA_(y) and AA_(z) are amino acids which together form a bridging bondselected from the group consisting of disulfide bonds, methylene bonds,sulfide/methylene, amide, and ester bonds;

X₁ is (H) or a peptide of 1 to 125 amino acid residues, or the desNH₂form thereof, or is a hydrophobic aliphatic, aromatic, or mixedaliphatic/aromatic organic group of from 6 to 20 carbon atoms; X₂ is abond or a peptide of 1-10 residues; provided the distance between theamino of AA₈ and a hydrophobic moiety occurring in X₁ AA_(y) X₂ isapproximately between 4.5 and 15 angstroms in an achievable3-dimensional conformation;

X₃ is a bond, or a peptide of 1 to 10 residues;

X₄ is (OH), NH₂, NHR' or NHR'R" wherein R' and R' are straight orbranched-chain alkyls (1-10C) wherein 1-2 nonadjacent C may be replacedby N, O, or S, or X₄ is a peptide of from 1 to 20 residues, includingthe carboxy-terminal amide or alkyl amide forms thereof or is absent.

In connection with the above definitions, however, the total ring sizemust be equivalent to that obtained by conventional disulfide bridgeformation between cysteines separated by 5 to 15 alpha-amino acids.Since the bridge in the invention compounds is not limited to cystine,the ring size is due to both the length of the primary chain and to thelength of the bridge.

In the foregoing compounds of the invention, one or more of the amidebackbone linkages between any adjacent amino acid residues mayoptionally be replaced by a linkage selected from the group consistingof --CH₂ NH--, --CH₂ --S--, --CH₂ CH₂ --, --CH═CH-- (cis and trans),--COCH₂ --, --CH(OH)CH₂ and --CH₂ SO--.

However, for those compounds of the invention wherein the bridge isformed by a disulfide between two cysteine residues, if AA₁₂ is not acovalent bond, and if X₂ is a tripeptide, X₃ cannot be a heptapeptide.

One or two of the residues in the peptides of the invention may bereplaced by the corresponding D isomer, in addition to, or instead of,AA₉ and AA₁₂.

Also provided in accordance with aspects of the invention arepharmaceutical compositions useful as natriuretics, diuretics,vasodilators and/or modulators of the renin-angiotensin-aldosteronesystem, which compositions containing the above-recited analog peptidecompounds, including their amides and esters, and the nontoxic additionsalts thereof, together with a pharmaceutically acceptable liquid, gelor solid carrier. Administration of therapeutically effective doses ofthese compositions can provide effective delivery of the above-recitedbiological activities to mammalian hosts.

Additional aspects of the present invention provide methods forproducing such compounds and compositions, and methods for using thecompounds and compositions as therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically outlines the classification of amino acids as usedherein.

FIG. 2 is a graph which depicts competitive displacement receptorbinding of compounds of the present invention using cultured bovineaortic smooth muscle (BASM) cells; and

FIG. 3 depicts the in vivo diuretic activities of selected compounds ofthe present invention in anesthetized rats, wherein

FIG. 3A portrays the diuretic activity of the analog peptide identifiedas AP25,

FIG. 3B portrays the diuretic activity of the analog peptide identifiedas AP20,

FIG. 3C portrays the diuretic activity of the analog peptide identifiedas AP21,

FIG. 3D portrays the diuretic activity of the analog peptide identifiedas AP37,

FIG. 3E portrays the diuretic activity of the analog peptide identifiedas AP101,

FIG. 3F portrays the diuretic activity of the analog peptide identifiedas AP319,

FIG. 3G portrays the diuretic activity of the analog peptide identifiedas AP324, and

FIG. 3H portrays the diuretic activity of the analog peptide identifiedas AP54.

FIG. 4 shows representative compounds of the invention.

FIG. 5 shows analogous compounds which are not included in theinvention.

MODES OF CARRYING OUT THE INVENTION

In accordance with the present invention, a class of novel analogs ofnative Atrial Natriuretic Peptide (ANP) compounds is provided which iscapable of exhibiting or modulating the natriuretic, diuretic and/orvasorelaxant activity of the native peptides in mammals in vivo.

The sequence of amino acid residues of the present synthetic analogcompounds, including the core pentapeptide, and preferred embodimentsthereof, are defined in terms of amino acids of certain characteristicsof particular subclasses.

Amino acid residues can be generally subclassified into four majorsubclasses as follows and as shown in FIG. 1.

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH.

Basic: The residue has a positive charge due to association with H ionat physiological pH and the residue is attracted by aqueous solution soas to seek the surface positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium atphysiological pH.

Neutral/nonpolar: The residues are not charged at physiological pH andthe residue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of"charged", a significant percentage (at least approximately 25%) of theindividual molecules are charged at physiological pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary, and, therefore, amino acids specifically contemplated bythe invention have been specifically classified as one or the other.Most amino acids not specifically named can be classified on the basisof known behavior.

Amino acid residues can be further subclassified as cyclic or noncyclicor aromatic or nonaromatic, self-explanatory classifications withrespect to the side chain substituent groups of the residues, and assmall or large. The residue is considered small if it contains a totalof 4 carbon atoms or less, inclusive of the carboxyl carbon. Smallresidues are, of course, always noncyclic and nonaromatic.

For the naturally occurring protein amino acids, subclassificationaccording to the foregoing scheme is as follows (see also FIG. 1)

Acidic: Aspartic acid and Glutamic acid;

Basic/noncyclic: Arginine, Lysine;

Basic/cyclic: Histidine;

Neutral/polar/small: Glycine, Serine and Cysteine;

Neutral/polar/large/nonaromatic: Threonine, Asparagine, Glutamine;

Neutral/polar/large/aromatic: Tyrosine;

Neutral/nonpolar/small: Alanine;

Neutral/nonpolar/large/nonaromatic: Valine, Isoleucine, Leucine,Methionine;

Neutral/nonpolar/large/aromatic: Phenylalanine, and Tryptophan.

The gene-encoded amino acid proline, although technically within thegroup neutral/nonpolar/large/cyclic and nonaromatic, is a special casedue to its known effects on the secondary conformation of peptidechains, and is not, therefore, included in this defined subclass.

Certain commonly encountered amino acids, which are not encoded by thegenetic code, include, for example, beta-alanine (beta-ala), or otheromega-amino acids, such as 3-amino propionic, 4-amino butyric and soforth, alpha-aminoisobutyric acid (Aib), sarcosine (Sar), ornithine(Orn), citrulline (Cit), t-butylalanine (t-BuA), t-butylglycine (t-BuG),N-methylisoleucine (N-MeIle), phenylglycine (Phg), and cyclohexylalanine(Cha), norleucine (Nle), cysteic acid (Cya) and methionine sulfoxide(MSO). These also fall conveniently into particular categories.

Based on the above definition,

Sar and beta-ala are neutral/nonpolar/small;

t-BuA, t-BuG, N-MeIle, Nle, and Cha areneutral/nonpolar/large/nonaromatic;

Orn is basic/noncyclic;

Cya is acidic;

Cit and MSO are neutral/polar/large/nonaromatic; and

Phg is neutral/nonpolar/large/aromatic

See, also, FIG. 1.

The various omega-amino acids are classified according to size asneutral/nonpolar/small (beta-ala, i.e., 3-aminopropionic,4-aminobutyric) or large (all others).

Other amino acid substitutions for those encoded in the gene can also beincluded in peptide compounds within the scope of the invention and canbe classified within this general scheme.

Each of the foregoing, and as shown in FIG. 1, is defined as a"subclass" herein, and, in general, a specific amino acid can bereplaced by "another member of its subclass."

The nomenclature used to describe ANP analog compounds of the presentinvention follows the conventional practice wherein the amino group isassumed to the left and the carboxy group to the right of each aminoacid in the peptide. In the formulas representing selected specificembodiments of the present invention, the amino- and carboxy-terminalgroups, although often not specifically shown, will be understood to bein the form they would assume at physiological pH values, unlessotherwise specified. Thus, the N-terminal-H₂ ⁺ and C-terminal-O⁻ atphysiological pH are understood to be present though not necessarilyspecified and shown, either in specific examples or in generic formulas.In the peptides shown, each encoded residue where appropriate isrepresented by a single letter designation, corresponding to the trivialname of the amino acid, in accordance with the following conventionallist:

    ______________________________________                                                       One-Letter                                                     Amino Acid     Symbol                                                         ______________________________________                                        Alanine        A                                                              Arginine       R                                                              Asparagine     N                                                              Aspartic acid  D                                                              Cysteine       C                                                              Glutamine      Q                                                              Glutamic acid  E                                                              Glycine        G                                                              Histidine      H                                                              Isoleucine     I                                                              Leucine        L                                                              Lysine         K                                                              Methionine     M                                                              Phenylalanine  F                                                              Proline        P                                                              Serine         S                                                              Threonine      T                                                              Tryptophan     W                                                              Tyrosine       Y                                                              Valine         V                                                              ______________________________________                                    

The amino acids not encoded genetically are abbreviated as indicatedabove.

In the specific peptides shown in the present application, the L-form ofany amino acid residue having an optical isomer is intended unlessotherwise expressly indicated by a dagger superscript, (), e.g., by thesymbol "(AA_(n) )". While the residues of the invention peptides arenormally in the natural L optical isomer form, one or two, preferablyone, amino acid in addition to as well as instead of AA₉ and/or AA₁₂,may be replaced with the optical isomer D form (including embodimentswhere AA₉ and AA₁₂ are both L).

Free functional groups, including those at the carboxy- oramino-terminus, can also be modified by amidation, acylation or othersubstitution, which can, for example, change the solubility of thecompounds without affecting their activity.

In particular, it has been discovered that carboxyl terminalamide-modified analogs of Atrial Natriuretic Peptides are particularlypotent and therefore preferred embodiments of the present invention. Ingeneral, the nitrogen atom of the amido group, covalently bound to thecarbonyl carbon, will be NH₂, --NHR', or NR'R", wherein R' and R" arestraight or branched chain alkyl or alkyl acyl of 1-10C, preferably1-6C, including these groups wherein 1-2 carbons are replaced bynitrogen, oxygen or sulfur atoms. Representatives of such amido groupsare: --NH₂, --NHCH₃, --N(CH₃)₂, --NHCH₂ CH₃, --NHC₆ H₅, --NH--CH₂--CH(CH₃)₂, --NH--CH₂ --CH(CH₃)CH₂ CH₃, --NHCH₂ CH₂ OH, --NHCH₂ OCH₂ CH₃and --N(CH₃)CH₂ CH₂ SCH₂ CH₃, among others.

In forming amidated analogs of the present invention, the analogcompounds can be synthesized directly, for example using Boc-AA_(x)-pMBHA-Resin or Boc-AA_(x) -BHA-Resin, wherein AA_(x) is the selectedcarboxy-terminal amino acid of the desired analog compound as describedin further detail below. Alternatively, the analog compounds of thepresent invention can be chemically or enzymatically amidated subsequentto peptide synthesis using means well known to the art, or prepared bystandard solution-phase peptide synthesis protocols.

PREFERRED EMBODIMENTS A. The Core Pentapeptide

The compounds of the invention all contain the pentapeptide coresequence:

    AA.sub.8 -AA.sub.9 -AA.sub.10 -AA.sub.11 -AA.sub.12

wherein

each of AA₈ and AA₁₁ is, independently:

a basic/noncyclic; or

a neutral/nonpolar/small; or

a neutral/polar/large/nonaromatic amino acid residue;

AA₉ is a neutral/nonpolar/nonaromatic amino acid residue in the D or Lconfiguration;

AA₁₀ is an acidic amino acid residue; and

AA₁₂ is a neutral/nonpolar/large/nonaromatic amino acid residue in the Dor L configuration, or is a covalent bond.

The most preferred sequence of this core is R(I/M)DRI, wherein allresidues are in the L configuration and the amino acid residuescontained within the parentheses are alternatives. Next in preferenceare those sequences wherein only one of the R(I/M)DRI residues has beensubstituted by an alternative residue within the above definitions.Preferred substitutions are:

For AA₈, instead of R: A, Q, N, K, L or Nle;

for AA₉, instead of I/M: V, V , L, L , I , M ,

t-BuA, t-BuG or Cha;

for A₁₀, instead of D: E or Cya;

for A₁₁, instead of R: A, Q, N, K, Orn, or Cit;

for A₁₂, instead of I: M, M , V, V , L, L , I ,

N-MeIle, t-BuA or a covalent bond.

Particularly preferred are those embodiments wherein this sequence isselected from the group consisting of:

    ______________________________________                                        A(I/M)DRI     RM DRI       R(I/M)DRL                                          K(I/M)DRI     RLDRI        R(I/M)DRM                                          Q(I/M)DRI     R(I/M)ERI    R(I/M)DRM                                          RVDRI         R(I/M)DKI    R(I/M)DRI                                          RI DRI        R(I/M)DQI    R(I/M)DRV                                          ______________________________________                                    

More than one alteration from the naturally occurring RIDRI or RMDRIsequence is within the scope of the invention, but less preferred.Particularly favored subsets of this group include those whereinglutamic replaces aspartic as AA₁₀, in addition to another substitution.

B. The Nature of the Ring; Preferred Embodiments for AAy and AAz

The cyclic disulfides included within the invention are directlyanalogous to the naturally occurring ANPs, which contain 17 amino acidresidue-member disulfide rings, inclusive of the two cysteine residueswhich provide the sulfhydryl groups for the formation of the disulfidebond. However, those embodiments of the compounds of the invention whichcontain the cyclic disulfide may contain either more or, much morepreferably less, than 17 amino acid residues in the cyclic structure.

As indicated, the cyclic compounds of the present invention can beprovided by bonding cysteine residues, or alternate amino acid residuesAA_(y) and AA_(z) with an equivalent bond or linking group such as, forexample, --CH₂ --CH₂ --. The replacement of a sulfhydryl group on thecysteine residue with an alternative group will effectively replace thecysteine residue with an alternative amino acid. For example, to replaceone sulfhydryl group with a --CH₂ -- group, the cysteine residues willbe replaced by the analogous alpha-aminobutyric acid. These cyclicanalog peptides can be formed, for example, in accordance with themethodology of Lebl, M. and V. J. Hruby, Tetrahedron Lett. (1984) 25:2067-2068, or by employing the procedure disclosed in U.S. Pat. No.4,161,521.

In addition to the disulfide and methylene bridges formed by twocysteines, by two alpha-amino butyric residues or by a composite ofthese, i.e.: ##STR1## ester or amide bridges may also be formed. Forexample, an ester bridge may involve the --OH of serine or threonine andcarboxyl of aspartic or glutamic, e.g., ##STR2## Similarly, an amide canconveniently be obtained using the side chains of lysine and aspartic orglutamic, e.g., ##STR3## Methods for synthesis of these bridges arefound in Schiller, P. W., et al, Biochem Biophys Res Comm (1985) 127:558-564; Schiller, P. W., et al, Int. J Peptide and Protein Res (1985)25: 171-177.

The amino acids which participate in the bridge, like the others whichcompose the peptide, may optionally be in the D-form, so long as no morethan one or two residues (aside from, or in lieu of, one of AA₉ andAA₁₂) are thus configured.

Thus, A_(y) can be any residue capable of forming a bridge--i.e., itcannot be Gly or an omega-amino straight chain acid unless it is in theN-terminal position. However, a methylene or substituted methylenecontribution to a methylene bridge or methylene/sulfide bridge may befurnished by alpha-aminobutyric, Val, Leu, Ile, and, for example,certain aromatic (hydrophobic) amino acids such as Phe. Disulfidelinkage atoms are contributed by cysteine or homocysteine; participantsin an ester or amide bridge are provided by Ser, Thr, Glu, Asp, Cya(this for a sulfonamide or sulfonate ester), Lys, and so forth. If X₁ isH, AA_(y) is the N-terminal amino acid and may participate in thebridge.

A_(z) is chosen from among amino acid residues capable of forming abridge to AA_(y). It is selected from the same general group as isA_(y), although, of course, if an amide or ester bridge is to be formedthe functional group furnished by A_(z) must be complementary to that ofA_(y). Also, if X₄ is OH, AA_(z) is the C terminal residue and the COOHof any residue in this position can participate in the bridge.

A preferred series wherein the carboxyl of the amide bridge is providedby the C-terminus include those wherein the C-terminus is Gly, beta-Ala,or an omega-amino acid of the formula H₂ N(CH₂)_(p) COOH, wherein p is3-6. The amino components are provided by the --NH₂ of lysine or ofornithine. In addition, the positions of the COOH donor and NH₂ donorsmay be reversed, provided these groups are available in the side chainsof the C-terminal and N-terminal residues.

C. Preferred Embodiments for X₁

X₁ can be, but need not be, a hydrophobic amino acid or otherhydrophobic group. When X₁ is a hydrophobic moiety, A_(y) X₂ is a groupwherein the intervening linkage can be conformed to a distance of aboutbetween or 4.5-15 angstroms. In this set of embodiments, particularlypreferred for X₁ are hydrophobic groups of 6-20 carbons, including,especially, phenylalanine or its des-NH₂ form and aromatic acetyl,butyryl, or propionyl derivatives, especially naphthyl, dibenzyl, andindolylacyl derivatives as set forth below.

Thus, one class of presently preferred organic substituent groups can berepresented by the general formula:

    R.sub.1 --CO--

wherein R₁ is an organic hydrophobic group. Included in this formula are2-substituted acetyl, 3-substituted propionyl, and 4-substituted butyrylgroups, wherein the substitutions to these groups include the generalclass of neutral, hydrophobic mono- and polycyclic aromatic or saturatedring systems. Other classes have the general formulas R₁ --O--CO-- andR₁ --O--. Representative examples of the preferred substituent groupsinclude: ##STR4## groups.

Other preferred embodiments for X₁ include peptides of 1-6 amino acidresidues or the des-NH₂ forms thereof. The X₁ peptide may advantageouslycontain a hydrophobic residue capable of being spaced 4.5-15 angstromsfrom AA₈. In the native sequence, the X₁ peptide is S-L-R-R-S-S or,putatively, some of the N-terminal deleted forms, such as L-R-R-S-S,R-R-S-S, and R-S-S. In these embodiments, too, one or more of theresidues may be replaced by another in its same class, e.g., R by K oranother basic amino acid, L by V or another neutral/large/nonpolar aminoacid, and S by G or another neutral/polar/small amino acid or by Ala. Inaddition, X₁ may be a peptide of further N-terminal deletions such asS-S, or S, also permitting substitution by comparable forms, or X₁ maysimply be hydrogen.

D. Preferred Embodiments for X₂

In the peptides of the invention, if the portion upstream from thepentapeptide core contains a hydrophobic moiety, it should be separatedfrom AA₈ over a space of about 4.5-15 angstroms. Therefore, the natureof the preferred forms for X₂ depend on whether or not a hydrophobicresidue occurs upstream (in X₁, as AA_(y), or in X₂). If a hydrophobicresidue does not occur, X₂ is preferably a bond or a peptide of 1-3amino acid residues wherein the residues are selected from the groupconsisting of Ala, Gly, and Ser. If X₁ contains a hydrophobe adjacentAy, X₂ is preferably a bond or a peptide of 1-2 amino acids, wherein theresidues are selected from neutral/polar/small or neutral/nonpolar/smallamino acids, in particular Gly, Ser, Ala, Aib, and Sar. Particularprefered forms include a bond, G, G-G, A-G, S-G, G-A, G-S, G-Aib, andG-Sar. If A_(y) is itself hydrophobic, prefered forms of X₂ furtherinclude tripeptides wherein the residues are selected fromneutral/polar/small and neutral/nonpolar/small, as above.

In the native cyclic forms, A_(y) is C and X₂ is a tripeptide of thesequence F-G-G. When X₂ includes F or other hydrophobic residue,preferred forms of X₂ are those wherein the C-terminal portion of the X₂peptide provides the required spacing of 4.5-15 angstroms. Especiallyfavored is G-G or that wherein the G-G dipeptide has one residuereplaced by Ser, Ala, Aib or Sar, or wherein only one residue of G (orits substitute) is present.

However, it is clear that in these cyclic forms, no upstreamhydrophobicity is necessary and the group X₁ AA_(y) X₂ may simply be apeptide extension.

E. Preferred Embodiments of X₃

X₃ is generally a peptide residue of such length that the ring willcontain 7-17 residues, including the AA_(y) and AA_(z) residues ifAA_(y) and AA_(z) are cysteines; or will form a ring of comparable sizeif they are not. Thus, if, for example, X₂ is a tripeptide, X₃ ispreferably a bond or a peptide containing 1-6 amino acid residues. X₃will preferably be a peptide which is a variant of the native sequenceG-A-Q-S-G-L-G or the truncated forms thereof, wherein one or more of theGly or Ser residues may be replaced by another neutral/polar/small aminoacid or by Ala, and Ala by another neutral/nonpolar/small amino acid orGly or Ser. Gln may be replaced by anotherneutral/polar/large/nonaromatic amino acid; and Leu may be replaced byanother neutral/nonpolar/large/nonaromatic amino acid.

The amino acid residues included in X₃ may in particular includeomega-amino forms. Thus, in a preferred embodiment, X₃ is a mono ordipeptide wherein one of the residues is of the formula --HN(CH₂)_(b)CO-- wherein b is 3-6.

Exemplary truncated forms also include

G-A-Q-S-G-L, A-Q-S-G-L-G, G-A-Q-S-G, Q-S-G-L-G, G-A-Q-S, S-G-L-G, G-A-Q,G-A-A, G-L-G, L-G, G-A, G and desX₃.

F. Preferred Embodiments of X₄

Preferred embodiments for X₄ are (OH), NH₂, NHR' wherein R' is straightor branched chain allyl of 1-10C wherein 1-2C can be replaced bynonadjacent N, O, or S, or a peptide of 1-5 particularly 1-2 amino acidresidues, and the amide or alkyl amide forms thereof, especiallyN-S-F-R-Y, N-S-F-R, N-S-F, N-S, or N and the amides thereof, andvariants wherein one or more residues is replaced by another of the sameclass or wherein X₄ is absent.

Preferred Compounds

Examples of preferred cyclic compounds include

    (2-naphthylacetyl)-C-G-R-I-D-R-I-G-A-C-NH.sub.2

    (2-naphthylacetyl)-C-G-R-I-D-R-I-C-NH.sub.2,

    (2-naphthylacetyl)-G-C-R-I-D-R-I-C-NH.sub.2,

    (2-naphthylacetyl)-G-C -R-I-D-R-I-G-C-NH.sub.2 and

    (2-naphthylacetyl)-G-C-R-I-D-R-I-G-A-C-NH.sub.2

where the Cys residues forming the ring can also be in the D form.

The preferred invention compounds also include cyclic compounds whichare not disulfides. Exemplary preferred compounds include

2-naphthylacetyl-K-G-R-I-D-R-I-G-G, which contains an amide linkagebetween the K amino sidechain and the G carboxyl;

2-naphthylacetyl-K-G-R-I-D-R-I-E-NH₂, which contains an amide linkagebetween the K amino sidechain and the E sidechain carboxyl;

2-naphthylacetyl-K-G-R-I-D-R-I-G-D-NH₂, which contains an amide linkagebetween the K amino sidechain and the D sidechain carboxyl.

H. Non-Peptide Linkages

In one embodiment of the invention, the amide linkages (--CO--NH--)within the core pentapeptide or those described above within Z₁ and/orZ₂ and/or Z₃ can be replaced with other types of linkages such as --CH₂NH--, --CH₂ --S--, --CH₂ CH₂ --, --CH═CH-- (cis and trans), --COCH₂ --,--C(OH)CH₂ -- and --CH₂ SO--, by methods known in the art. The followingreferences describe preparation of peptide analogs which include thesealternative-linking moieties: Spatola, A. F. Vega Data, (March 1983),Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review);Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids Peptidesand Proteins", B Weinstein, eds., Marcel Dekker, New York, p. 267 (1983)(general review); Morley, J. S. Trends Pharm Sci (1980) pp. 463-468(general review); Hudson, D. et al Int J Pept Prot Res (1979) 14:177-185 (--CH₂ NH-- , --CH₂ CH₂ --); Spatola, A. F. et al, Life Sci(1986) 38: 1243-1249 (--CH₂ --S); Hann, M. M. J. Chem Soc Perkin Trans I(1982) 307-314 (--CH--CH--, cis and trans); Almquist, R. G., et al, J.Med Chem (1980) 23: 1392-1398 (--COCH₂ --); Jennings-White, C. et alTetrahedron Lett (1982) 23: 2533 (--COCH₂ --); Szelke, M., et al,European Appln EP 45665 (1982) CA: 97: 39?05 (1982) (--C(OH)CH₂ --);Holladay, M. W. et al Tetrahedon Lett (1983) 24: 4401-4404 (--C(OH)CH₂--); and Hruby, V. J. Life Sci (1982) 31: 189-199 (--CH₂ --S--).

I. Synthesis

Compounds within the scope of the present invention can be synthesizedchemically by means well known in the art such as, e.g., solid-phasepeptide synthesis. The synthesis is commenced from the carboxy-terminalend of the peptide using an alpha-amino protected amino acid.t-Butyloxycarbonyl (Boc) protective groups can be used for all aminogroups even though other protective groups are suitable. For example,Boc-Asn-OH, Boc-Ser-OH, Boc-Phe-OH, Boc-Arg-OH or Boc-Tyr-OH (i.e.,selected ANP analog carboxy-terminal amino acids) can be esterified tochloromethylated polystyrene resin supports. The polystyrene resinsupport is preferably a copolymer of styrene with about 0.5 to 2%divinyl benzene as a cross-linking agent which causes the polystyrenepolymer to be completely insoluble in certain organic solvents. SeeStewart, et al, Solid-Phase Peptide Synthesis (1969) W. H. Freeman Co.,San Francisco and Merrifield, J Am Chem Soc (1963) 85: 2149-2154. Theseand other methods of peptide synthesis are also exemplified by U.S. Pat.Nos. 3,862,925, 3,842,067, 3,972,859, and 4,105,602.

The synthesis may use manual techniques or automatically employing, forexample, an Applied BioSystems 430A Peptide Synthesizer (Foster City,Calif.) or a Biosearch SAM II automatic peptide synthesizer (Biosearch,Inc. San Rafael, Calf.), following the instructions provided in theinstruction manual supplied by the manufacturer.

It will be readily appreciated by those having ordinary skill in the artof peptide synthesis that the intermediates which are constructed inaccordance with the present disclosure during the course of synthesizingthe present analog compounds are themselves novel and useful compoundsand are thus within the scope of the invention.

Alternatively, selected compounds of the present invention can beproduced by expression of recombinant DNA constructs prepared inaccordance with well-known methods. Such production can be desirable toprovide large quantities or alternative embodiments of such compounds.Since the peptide sequences are relatively short, recombinant productionis facilitated.

J. Administration and Use

Compounds of the present invention are shown to have natriuretic,diuretic and hypotensive activity in the intact mammal, and may possessvasorelaxant activity or inhibit the release of aldosterone and renin.

Thus these compounds, and compositions containing them, can find use astherapeutic agents in the treatment of various edematous states such as,for example, congestive heart failure, nephrotic syndrome and hepaticcirrhosis, in addition to hypertension and renal failure due toineffective renal perfusion or reduced glomerular filtration rate.

Thus the present invention also provides compositions containing aneffective amount of compounds of the present invention, including thenontoxic addition salts, amides and esters thereof, which may, alone,serve to provide the above-recited therapeutic benefits. Suchcompositions can also be provided together with physiologicallytolerable liquid, gel or solid diluents, adjuvants and excipients.

These compounds and compositions can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapuetic efficacy will range from about 0.01 to1000 mcg/kg, more usually 0.1 to 1000 mcg/kg of the host body weight.Alternatively, dosages within these ranges can be administered byconstant infusion over an extended period of time, usually exceeding 24hours, until the desired therapeutic benefits have been obtained.

Typically, such compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared. Thepreparation may also be emulsified. The active ingredient is often mixedwith diluents or excipients which are physiologically tolerable andcompatible with the active ingredient. Suitable diluents and excipientsare, for example, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired the compositions maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, stabilizing or pH-buffering agents, and the like.

The compositions are conventionally administered parenterally, byinjection, for example, either subcutaneously or intravenously.Additional formulations which are suitable for other modes ofadministration include suppositories, intranasal aerosols, and, in somecases, oral formulations. For suppositories, traditional binders andexcipients may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10% preferably 1%-2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained-release formulations, or powders, and contain10%-95% of active ingredient, preferably 25%-70%.

The peptide compounds may be formulated into the compositions as neutralor salt forms. Pharmaceutically acceptable nontoxic salts include theacid addition salts (formed with the free amino groups) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or organic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups maybe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

In addition to the compounds of the present invention which displaynatriuretic, diuretic or vasorelaxant activity, compounds of the presentinvention can also be employed as intermediates in the synthesis of suchuseful compounds. Alternatively, by appropriate selection, compounds ofthe present invention whose activity levels are reduced or eliminatedentirely can serve to modulate the activity of other diuretic,natriuretic or vasorelaxant compounds, including compounds outside thescope of the present invention, by, for example, binding to alternatereceptors, stimulating receptor turnover, or providing alternatesubstrates for degradative enzyme or receptor activity and thusinhibiting these enzymes or receptors. When employed in this manner,such compounds can be delivered as admixtures with other activecompounds or can be delivered separately, for example, in their owncarriers.

Compounds of the present invention can also be used for preparingantisera for use in immunoassays employing labeled reagents, usuallyantibodies. Conveniently, the polypeptides can be conjugated to anantigenicity-conferring carrier, if necessary, by means of dialdehydes,carbodiimide or using commercially available linkers. These compoundsand immunologic reagents may be labeled with a variety of labels such aschromophores, fluorophores such as, e.g., fluorescein or rhodamine,radioisotopes such as ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³ H, or magnetizedparticles, by means well known in the art.

These labeled compounds and reagents, or labeled reagents capable ofrecognizing and specifically binding to them, can find use as, e.g.,diagnostic reagents. Samples derived from biological specimens can beassayed for the presence or amount of substances having a commonantigenic determinant with compounds of the present invention. Inaddition, monoclonal antibodies can be prepared by methods known in theart, which antibodies can find therapeutic use, e.g., to neutralizeoverproduction of immunologically related compounds in vivo.

The following examples are provided by way of illustration, rather thanimplying any limitation of the subject invention.

EXAMPLES

In the experimental disclosure which follows, the amino acid sequence ofchemically synthesized ANP analog compounds are numbered from theamino-terminal arginine residue corresponding to the arginine residuefound at position 1 in the native rat-derived Atrial Natriuretic Peptidesequence disclosed in Atlas, S., et al, Nature (1984) 309: 717-719.

I. CHEMICAL SYNTHESIS OF ATRIAL NATRIURETIC PEPTIDE ANALOG COMPOUNDS A.SYNTHESIS PROCEDURES

Compounds of the present invention were synthesized by solid-phasetechniques performed manually or, alternatively, on an AppliedBioSystems 430A Peptide Synthesizer (Foster City, Calif.) or a BiosearchSam II automated peptide synthesizer (Biosearch, San Rafael, Calif.)using t-Boc amino acids in accordance with the instructions of themanufacturer.

PROCEDURE A Preparation of Boc-AA₁ . . . AA_(n-1) -AA_(n) -ResinHydroxymethyl Polystyrene Ester

One gram of selected Boc-AA_(n) -O-Polystyrene-Resin (0.2-0.6 mmole/gresin) (obtainable from, e.g., Peninsula Labs, Inc.) is treatedaccording to schedule A for incorporation of the Boc-AA_(n-1) -OH.

    ______________________________________                                        Schedule A                                                                    ______________________________________                                        (1)     Wash 3x with dichloromethane (CH.sub.2 Cl.sub.2);                     (2)     Treat for 1 min. with TFA:CH.sub.2 Cl.sub.2 :ethane                           dithiol (EDT) (45:50:5 by volume);                                    (3)     Treat for 20 min. with TFA:CH.sub.2 Cl.sub.2 :EDT                             (45:50:5) by volume;                                                  (4)     Wash 3x with CH.sub.2 Cl.sub.2 ;                                      (5)     Treat 2x for 1 min. 10% (V/V)                                                 Diisoprophylethylamine (DIPEA) in CH.sub.2 Cl.sub.2 ;                 (6)     Wash 2x with CH.sub.2 Cl.sub.2 ;                                      (7)     Wash 2x with methanol (MeOH);                                         (8)     Repeat (5-7) once;                                                    (9)     Wash 3x with CH.sub.2 Cl.sub.2 ;                                      (10)    Add 1-6 equivalents of preformed sym-                                         metrical anhydride of the suitably                                            protected Boc-amino acid dissolved in                                         CH.sub.2 Cl.sub.2 or dimethyl formamide (DMF)/CH.sub.2 Cl.sub.2               (50:50 volume), (Boc-N-OH, Boc-Q-OH and                                       Boc-R(TOS)-OH were coupled as active esters                                   using N-hydroxybenzotriazole);                                        (11)    Wash 2x with CH.sub.2 Cl.sub.2 ;                                      (12)    Wash 2x with 10% DIPEA;                                               (13)    Wash 2x with CH.sub.2 Cl.sub.2 ;                                      (14)    Wash 2x with MeOH;                                                    (15)    Wash 2x with CH.sub.2 Cl.sub.2 ;                                      (16)    Repeat steps (11-15) once;                                            (17)    Test by ninhydrin reaction according to                                       Kaiser et al., Anal. Biochem. 34:595                                          (l970). If the coupling reaction was in-                                      complete, repeat steps (10-16) or,                                            alternatively, cap synthesis using N-acetyl                                   imidazole (0.30 M in DMF) or an excess of                                     acetic anhydride in CH.sub.2 Cl.sub.2.                                ______________________________________                                    

PROCEDURE B Preparation of Boc-AA_(n) -p-Methylbenzhydrylamine resin

The selected Boc-AA_(n) -OH is attached to a p-Methylbenzhydrylamine(pMBHA) resin via N,N'-dicyclohexylcarbodiimide, as described below.

    ______________________________________                                        Schedule B                                                                    ______________________________________                                        (1)     Wash the pMBHA HCl resin;                                             (2)     Wash the resin 2x with 10% (V/V) DIPEA in                                     CH.sub.2 Cl.sub.2 ;                                                   (3)     Wash 2x with CH.sub.2 Cl.sub.2 ;                                      (4)     Wash 2x with MeOH;                                                    (5)     Wash 2x with Ch.sub.2 Cl.sub.2 ;                                      (6)     Add 1-6 equivalents of preformed sym-                                         metrical anhydride of the suitably                                            protected Boc-amino acid dissolved in                                         CH.sub.2 Cl.sub.2, with reaction time of 0.5-24                       ______________________________________                                                hrs.                                                              

Unreacted amino groups are acetylated with 0.30M N-acetylimidazole:DMF,or acetic anhydride:CH₂ Cl₂. The following examples demonstrate thechemical synthesis of representative analog ANP compounds (identified asAP#) which illustrate certain aspects of the present invention.

EXAMPLE 1 *AP1 R-S-S-C-F-G-G-R-I-D-R-I-G-A-Q-S-G-C-N-S-F-R-Y

One gm of Boc-Tyr(2BrZ)-O-Resin (0.54 meq/gm, Peninsula Labs Inc.,Belmont, CA) was subjected to procedure A with the required sequence ofamino acids (introduced in order as Boc-Arg(Tos)-OH, Boc-Phe-OH,Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Cys(4-CH₃ Bzl)-OH, Boc-Gly-OH,Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Ala-OH, Boc-Gly-OH, Boc-Ile-OH 1/2H₂ O,Boc-Arg(Tos)-OH, Boc-Asp(OBzl)-OH, Boc-Ile-OH 1/2H₂ O, Boc-Arg(Tos)-OH,Boc-Gly-OH, Boc-Gly-OH, Boc-Phe-OH, Boc-Cys(4-CH₃ Bzl)-OH,Boc-Ser-(Bzl)-OH, Boc-Ser-(Bzl)-OH, Boc-Arg(Tos)-OH). The protectedpeptidyl resin was treated with TFA:CH₂ Cl₂ :EDT (45:50:5 v/v/v) for 1min., then 20 min. and washed 3 times with CH₂ Cl₂ and 2 times with MeOHto give the TFA salt of the peptidyl resin, and dried in vacuo.

The peptidyl resin was then suspended in anhydrous hydrogen fluoride(HF) containing 10% anisole, 2% ethyl methyl sulfide for 30 min. at -10°C., and 30 min. at 0° C. The HF was removed by evaporation under vacuumand the peptide/resin mixture was suspended in diethyl ether. Thepeptide/resin mixture was washed twice with diethyl ether, once withchloroform, once with diethyl ether, once with chloroform and once withdiethyl ether. The peptide was extracted from the mixture with 2.0Macetic acid, diluted with H₂ O and lyophilized, to give the unoxidizedsulfhydryl peptide.

The crude peptide was dissolved in deoxygenated 0.01M ammonium acetate(NH₄ OAc), pH 7.9, to 0.5 mg/ml and then oxidized by dropwise additionof a slight excess of 0.01M potassium ferricyanide (KCN) solution,stirred 20 minutes and adjusted to pH 5 with acetic acid. The peptidesolution was treated with DOWEX AG3X4 anion exchange resin, filtered,diluted with H₂ O and lyophilized to give the crude cyclized peptide.

Purification of the peptide was achieved by desalting on Sephadex® G-25F(Pharmacia Fine Chemicals) using 0.5M AcOH as eluant, followed by ionexchange chromatography on CM-Sepharose® (Pharmacia Fine Chemicals) orCM-cellulose (Whatman) using an elution gradient generated by additionof 300 mM NH₄ OAc, pH 6.5, to a solution of 10 mM NH₄ OAc, pH 4.5.Fractions were collected having a minimum 97% purity, as judged byreversed phase HPLC, then pooled and lyophilized from H₂ O several timesto yield the purified AP1 acetate salt.

EXAMPLE 2 *AP2 R-S-S-C-G-R-I-D-R-I-G-A-Q-S-G-C-N-S-F-R-Y

One gm of Boc-Tyr(2BrZ)-O-Resin (0.54 meq/gm, Peninsula Labs Inc.,Belmont, CA) was subjected to procedure A with the required sequence ofamino acids (introduced in order as Boc-Arg(Tos)-OH, Boc-Phe-OH,Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Cys(4-CH₃ Bzl)-OH, Boc-Gly-OH,Boc-Ser(Bzl)-OH, Boc-Gln-OH, Boc-Ala-Oh, Boc-Gly-OH, Boc-Ile-OH 1/2H₂ O,Boc-Arg(Tos)-OH, Boc-Asp(OBzl)-OH, Boc-Ile-OH 1/2H₂ O, Boc-Arg(Tos)-OH,Boc-Gly-OH, Boc-Cys(4CH₃ Bzl)-OH, Boc-Ser(Bzl)-OH, Boc-Ser(Bzl)-OH,Boc-Arg(Tos)-OH). The protected peptidyl resin was treated with TFA:CH₂Cl₂ :EDT (45:50:5 v/v/v) for 1 min., then 20 min. and washed 3 timeswith CH₂ Cl₂, 2 times with MeOH and dried in vacuo to give the TFA saltof the peptidyl resin.

The peptidyl resin was then suspended in anhydrous HF containing 10%anisole, 2% ethyl methyl sulfide for 30 min. at -10° C. and 30 min. at0° C. The HF was removed by evaporation under vacuum and thepeptide/resin mixture was suspended in diethyl ether. The peptide/resinmixture was washed twice with diethyl ether, twice with chloroform, andtwice with diethyl ether. The peptide was extracted with 2.0M aceticacid and lyophilized, to give the unoxidized sulfhydryl peptide.

The crude peptide was dissolved in deoxygenated 0.01M NH₄ OAc, pH 7.9,to 0.5 mg/ml and then oxidized by dropwise addition of a slight excessof 0.01M KCN solution, stirred for 20 minutes and adjusted to pH 5 withacetic acid. The peptide solution was treated with DOWEX AG3X4 anionexchange resin, filtered, diluted with H₂ O and lyophilized to give thecrude cyclized peptide.

Purification of the peptide was achieved by desalting on Sephadex® G-25Fusing 0.5M AcOH as eluant, followed by ion exchange chromatography onCM-Sepharose® or CM-cellulose (Whatman) using an elution gradientgenerated by addition of 300 mM NH₄ OAc, pH 6.5, to a solution of 10 mMNH₄ OAc, pH 4.5. Fractions were collected having a minimum 97% purity,as judged by reversed phase HPLC, then pooled and lyophilized from H₂ Oseveral times to yield the purified AP2 acetate salt.

EXAMPLE 3 *AP3 R-S-S-C-F-G-G-R-I-D-R-I-G-A-Q-S-C-N-S-F-R-Y

One gm of Boc-Tyr(2BrZ)-O-Resin (0.54 meq/gm, Peninsula Labs Inc.,Belmont, CA) was subjected to procedure A with the required sequence ofamino acids (introduced in order as Boc-Arg(Tos)-OH, Boc-Phe-OH,Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Cys(4-CH₃ Bzl)-OH, Boc-Ser(Bzl)-OH,Boc-Gln-OH, Boc--Ala-OH, Boc-Gly-OH, Boc-Ile-OH 1/2H₂ O,Boc-Arg(Tos)-OH, Boc-Asp(OBzl)-OH, Boc-Ile-OH 1/2/H₂ O, Boc-Arg(Tos)-OH,Boc-Gly-OH, Boc-Gly-OH, Boc-Phe-OH, Boc-Cys(4CH₃ Bzl)-OH,Boc-Ser(Bzl)-OH, Boc-Ser(Bzl)-OH, Boc-Arg(Tos)-OH). The protectedpeptidyl resin was treated with TFA:CH₂ Cl₂ :EDT (45:50:5 v/v/v) for 1min., then 20 min. and washed 3 times with CH₂ Cl₂ and twice with MeOHto give the TFA salt of the peptidyl resin and dried in vacuo.

The peptidyl resin was then suspended in anhydrous HF containing 10%anisole, 2% ethyl methyl sulfide for 30 min. at -10° C. and for 30 min.at 0° C. The HF was removed by evaporation under vacuum and thepeptide/resin mixture was suspended in diethyl ether. The peptide/resinmixture was washed twice with diethyl ether, once with chloroform, oncewith diethyl ether, once with chloroform and once again with diethylether. The peptide was extracted from the mixture with 2.0M acetic acid,diluted with H₂ O and lyophilized, to give the unoxidized sulfhydrylpeptide.

The crude peptide was dissolved in deoxygenated 0.01M NH₄ OAc, pH 8, to0.5 mg/ml and then oxidized by dropwise addition of a slight excess of0.01M KCN solution, stirred 20 minutes and adjusted to pH 5 with aceticacid. The peptide solution was treated with DOWEX AG3X4 anion exchangeresin, filtered, diluted with H₂ O and lyophilized to give the crudecyclized peptide.

Purification of the peptide was achieved by desalting on Sephadex® G-25Fusing 0.5M AcOH as eluant, followed by ion exchange chromatography onCM-Sepharose® or CM-cellulose (Whatman) using an elution gradientgenerated by addition of 300 mM NH₄ OAc to a solution of 10 mM NH₄ OAc,pH 4.5. Fractions were collected having a minimum 97% purity, as judgedby reversed phase HPLC, then pooled and lyophilized from H₂ O severaltimes to yield the purified AP3 acetate salt.

EXAMPLE 4 *AP4 R-S-S-C-F-G-G-R-I-D-R-I-G-A-C-N-S-F-NH₂

One gm of Boc-Phe-pMBHA resin, obtained using schedule B, was subjectedto procedure A with the required sequence of amino acids (introduced inorder as Boc-Ser(Bzl)-OH, Boc-Asn-OH, Boc-Cys(4-CH₃ Bzl)-OH, Boc-Ala-OH,Boc-Gly-OH, Boc-Ile-OH.1/2H₂ O, Boc-Arg(Tos)-OH, Boc-Asp(OBzl)-OH,Boc-Ile-OH.1/2H₂ O, Boc-Arg(Tos)-OH, Boc-Gly-OH, Boc-Gly-OH, Boc-Phe-OH,Boc-Cys(4-CH₃ Bzl)-OH, Boc-Ser(Bzl)-OH, Boc-Ser(Bzl)-OH,Boc-Arg(Tos)-OH). The peptidyl resin was then suspended in anhydrous HFcontaining 10% anisole, 2% ethyl methyl sulfide for 30 min. at 10° C.and for 30 min. at 0° C. The HF was removed by evaporation under vacuumand the peptide/resin mixture was suspended in diethyl ether. Thepeptide/resin mixture was washed twice with diethyl ether, once withchloroform, once with diethyl ether, once with chloroform and once againwith diethyl ether. The peptide was extracted from the mixture with 2.0Macetic acid, diluted with H₂ O and lyophilized, to give the unoxidizedsulfhydryl peptide.

The crude peptide was dissolved in deoxygenated 0.01M NH₄ OAc, pH 8, to0.5 mg/ml and then oxidized by dropwise addition of a slight excess of0.01M KCN, stirred for 20 minutes and adjusted to pH 5 with acetic acid.The peptide solution was treated with DOWEX AG3X4 anion exchange resin,filtered, diluted with H₂ O and lyophilized to give the crude cyclizedpeptide.

Purification of the peptide was achieved by desalting on Sephadex® G-25Fusing 0.5M AcOH as eluant, followed by ion exchange chromatography onCM-Sepharose® or CM-cellulose (Whatman) using an elution gradientgenerated by addition of 300 mM NH₄ OAc to a solution of 10 mM NH₄ OAc,pH 4.5. Fractions were collected having a minimum 97% purity, as judgedby reversed phase HPLC, then pooled and lyophilized from H₂ O severaltimes to yield the purified AP4 acetate salt.

The following examples demonstrate the chemical synthesis ofrepresentative organic substituent group modified analog peptidecompounds (identified as AP#) which illustrate certain aspects of thepresent invention.

EXAMPLE 306 * AP306 (2-Naphthylacetyl)-G-G-R-I-D-R-I-G-A-NH₂

One gm of Boc-Ala-pMBHA resin (0.4 meq/gm), obtained using schedule B,was subjected to procedure A with the required sequence of amino acidsand Amino-terminal substituent group (introduced in order as Boc-Gly-OH,Boc-Ile-OH 1/2H₂ O, Boc-Arg(Tos)-OH, Boc-Asp(OBzl)-OH, Boc-Ile-OH 1/2H₂O, Boc-Arg(Tos)-OH, Boc-Gly-OH, Boc-Gly-OH, 2-Naphthylacetic acid). Theprotected peptidyl resin was washed 3 times with CH₂ Cl₂ and 3 timeswith MeOH and dried in vacuo.

The peptidyl resin was then suspended in anhydrous HF containing 10%anisole, 2% ethyl methyl sulfide for 30 min. at -10° C. and for 30 min.at 0° C. The HF was removed by evaporation under vacuum and thepeptide/resin mixture was suspended in ethyl ether. After transfer to afritted funnel, the peptide/resin mixture was washed twice with ethylether, once with chloroform, once with ethyl ether, once with chloroformand once again with ethyl ether. The peptide was then extracted from themixture with 2.0M acetic acid, diluted with H₂ O and lyophilized.

Purification of the peptide was achieved by ion exchange chromatographyon CM-Sepharose® (Pharmacia) using an elution gradient generated byaddition of 100 mM NH₄ OAc, pH 6.5, to a solution of 10 mM NH₄ OAc, pH4.5. Fractions were monitored at 254 nm and analyzed by reversed phaseHPLC. Fractions having a minimum 97% purity were pooled and lyophilizedfrom H₂ O several times to yield the purified AP306 acetate salt.

EXAMPLE 307 * AP307 (2-Naphthoxyacetyl)-NH(CH₂)₄ CO-R-I-D-R-I-NH₂

One gm of Boc-Ile-pMBHA resin (0.4 meq/gm), obtained using schedule B,was subjected to procedure A with the required sequence of amino acidsand Amino-terminal substituent group (introduced in order asBoc-Arg(Tos)-OH, Boc-Asp(OBzl)-OH, Boc-Ile-OH 1/2H₂ O, Boc-Arg(Tos)-OH,Boc-NH(CH₂)₄ COOH, 2-Naphthoxyacetic acid). The protected peptidyl resinwas washed three times with CH₂ Cl₂ and three times with MeOH and driedin vacuo.

The peptidyl resin was then suspended in anhydrous HF containing 10%anisole, 2% ethyl methyl sulfide for 30 min. at -10° C. and for 30 min.at 0° C. The HF was removed by evaporation under vacuum and thepepetide/resin mixture was suspended in ethyl ether. After transfer to afritted funnel, the peptide/resin mixture was washed twice with ethylether, once with chloroform, once with ethyl ether, once with chloroformand once again with ethyl ether. The peptide was then extracted from themixture with 2.0M acetic acid, diluted with H₂ O and lyophilized.

Purification of the peptide was achieved by ion exchange chromatographyon CM-Sepharose® (pharmacia) using an elution gradient generated byaddition of 100 mM NH₄ OAc, pH 6.5, to a solution of 10 mM NH₄ OAc, pH4.5. Fractions were monitored at 254 nm and analyzed by reversed-phaseHPLC. Fractions having a minimum 97% purity were pooled and lyophilizedfrom H₂ O several times to yield the purified AP307 acetate salt.

Following the procedures outlined in Examples 1-6 (to produce analogpeptides AP1-4 and 306 and 307) with appropriate modification, the ANPanalogs shown in FIG. 4 are synthesized. If the compound contains two"C" residues, the ring formed by a disulfide is implied; otherwise thering is shown. Part A of the table shows compounds synthesized in amanner analogous to Examples 1-4; part B to Examples 306 and 307. Inpart B, the following abbreviations are used.

AA=Adamantylacetyl

BPA=Biphenylacetyl

CHA=Cyclohexylacetyl

DBA=Dibenzylacetyl

DPP=Diphenylpropionyl

IB=Indolebutyryl

IP=Indoleproprionyl

NA=Naphthylacetyl

NL=Naphthyl

NM=Naphthylmethyl

NO=Naphthoxy

NOA=Naphthoxyacetyl

NTA=Naphthylthioacetyl

NYL=Naphthoyl

POP=Phenoxypropionyl

TPP=Triphenylpropionyl

MeONAP=Methoxynaphthylpropionyl

Starred compounds were verified as to sequence by sequence analysis.

II. BIOLOGICAL TESTING

Biological activity data for selected analog Atrial Natriuretic Peptides(ANPs) which were synthesized as disclosed above are presented below asbiochemical, isolated tissue and whole mammal bioassays.

Without intending to be bound by any theory, it is believed that theactivity of the ANP analog compounds of the invention is due to theiraffinity for receptors in the kidney and other sites which areresponsible for influencing the clearance of the endogenous ANPs. Thefollowing in vitro biological data show that the analog compounds of theinvention compete with an iodinated native ANP molecule for binding toreceptors from cultured bovine aortic smooth muscle (BASM) cells, andbovine endothelial (BAE) cells. This competition is, evidently,diagnostic for the binding to the relevant clearance receptors. Thiscorrelation is confirmed by in vitro data which demonstrate that analogsactive in the competitive binding assay (subsection A) are able to causediuresis and natriuresis in anesthetized rats and dogs and to lowerblood pressure in anesthetized rats (subsection B). However, the analogsdo not cause diuresis or natriuresis in isolated kidney, but potentiatethe effect of "natural" ANP in the isolated tissue (subsection C). Inaddition, the analogs of the invention show reduced cyclic GMP activity,an activity which is a hallmark of the direct biological function ofANP.

The results below demonstrate that a wide range of peptides within thescope of the invention test positive in an in vitro assay, which is thenconfirmed, using representative peptides, as a model fornatriuretic/diuretic and vasodilator activity in vivo. It is alsopostulated by the inventors that many if not all of the peptides andpeptide analogs disclosed herein will have oral activity as well.

A. RECEPTOR BINDING ASSAYS

Specific ANP receptor sites have been identified on target tissues, suchas kidney, adrenal, blood vessels, and cultured cells. Napier, M. A., etal, Proc Nat Acad Sci USA (1984) 81: 5946-5940; DeLean, A., et al,Endocrinology (1984) 115: 1636-1638; Schenk, D. B., et al, BiochemBiophys Res Comm (1985) 127: 433-442. Since the binding of ANP or ANPanalogs to these specific receptor sites is presumptively a prerequisiteof biological activity, binding of ANP analogs to these receptors isconsidered predictive of biological activity.

An assay has been developed, generally in accordance with the disclosureof Schenk, supra, and Scarborough, R. M., et al, J Biol Chem (1986) 261:12960-12964, which evaluates the ability of ANP analogs to compete witha labeled native ANP for binding to cultured BASM and BAE cells. Thisnative ANP, having the amino acid sequence: ##STR5## was iodinated onthe carboxy-terminal Y residue and is identified as (¹²⁵I)-rANP(126-150). Analogous "competitive displacement" receptor bindingassays are considered commonplace in the art for examining specificligand-receptor interactions. An example of the results of thisANP-receptor binding assay is presented in FIG. 2.

In this assay, 0.5 nM (¹²⁵ I)-rANP(126-150) was incubated in eachindividual sample of BASM cells in the presence of varying amounts ofunlabeled rANP(126-150) or a compound of the present invention havingthe amino acid sequence:

    AP25 R-S-S-C-R-I-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y

    AP37 C-F-G-G-R-I-D-R-I-G-A-C-NH.sub.2

    AP101 A-F-G-G-R-I-D-R-I-G-A-NH.sub.2 or

    AP132 (desNH.sub.2 -F)-G-G-R-I-D-R-I-NH.sub.2

As shown in FIG. 2, increasing concentrations of rANP(126-150), oranalog peptides AP25, AP37, AP101 or AP132, effectively prevent (¹²⁵I)-rANP(126-150) binding to BASM cell-associated receptors. Theconcentration of unlabeled peptide at which 50% of maximal (¹²⁵I)-rANP(126-150) binding is displaced is called Ki(app) and reflectsreceptor-binding affinity. Therefore, hypothetical peptide A, withKi(app)=100 nM, displays substantially weaker interaction with areceptor than hypothetical peptide B with a Ki(app)=10 nM. Assumingthese ANP analogs act at one or more ANP receptor sites, then increasedreceptor affinity should reflect increased biological potency.

Tables 1A-1F present data which compare the concentrations at whichanalog compounds of the present invention displace (¹²⁵ I)-rANP(126-150)binding from specific receptor sites on BASM or BAE cells. As will beshown below, these data correlate with in vivo activity charactericticof native ANP.

                                      TABLE 1A                                    __________________________________________________________________________    Peptide Sequence                Ki(app)(nM)                                   __________________________________________________________________________    rANP(126-150)                                                                         R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               7.26                                                  A--Q--S--G--L--G--C--N--S--F--R--Y                                    AP23    R--S--S--C--G--G--R--I--D--R--I--G--                                                                  7.76                                                  A--Q--S--G--L--G--C--N--S--F--R--Y                                    AP24    R--S--S--C--G--R--I--D--R--I--G--                                                                     25.2                                                  A--Q--S--G--L--G--C--N--S--F--R--Y                                    AP25    R--S--S--C--R--I--D--R--I--G--                                                                        8.62                                                  A--Q--S--G--L--G--C--N--S--F--R--Y                                    AP54    R--S--S--C--I--D--R--I--G--                                                                           >200                                                  A--Q--S--G--L--G--C--N--S--F--R--Y                                    __________________________________________________________________________

Table 1A compares receptor-binding affinity (Ki(app)) of rANP(126-150)with Ki(app) of various cyclic analog peptides. Also included, as anegative control, is AP54 which lacks a residue of the requisitepentapeptide. As shown, it does not bind to the receptor.

The data in Table 1A demonstrate that in these cyclic forms, it is notnecessary to include a hydrophobic residue proximal to the pentapeptidecore. However, as shown below, it may be advantageous to do so. Althoughanalog peptide AP24, where X₂ is G, has a weaker apparent affinity,analog peptide AP23, where X₂ is G-G and analog peptide AP25, where X₂is desX₂, exhibit equivalent receptor affinities to that ofrANP(126-150). Rings having as few as 14 amino acids are active. Asexpected, receptor binding capacity is substantially diminished forAP54, and AP54 should exhibit weaker biological activity if the Ki(app)correlates to activity. This is confirmed in Section B below; compoundsof the invention shown in Table 1A exhibit in vivo activity, whereas thenegative control, AP54, does not.

The correlation between Ki (app) and biological activity in vivo isfurther supported for certain linear analogs as set forth in Ser. No.138,893 filed 24 Dec. 1987 and incorporated herein by reference. Table1B shows data from this predictive in vitro test for compoundscontaining a hydrophobic residue within the required distance of AA₈,and within the ring, and wherein the ring size is as small as 10 aminoacids, including the two cysteines:

                                      TABLE 1B                                    __________________________________________________________________________    Peptide Sequence                Ki(app)(nM)                                   __________________________________________________________________________    rANP(126-150)                                                                         R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               7.26                                                  A--Q--S--G--L--G--C--N--S--F--R--Y                                    AP1     R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               6.88                                                  A--Q--S--G--C--N--S--F--R--Y                                          AP3     R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               9.l2                                                  A--Q--S--C--N--S--F--R--Y                                             AP4     R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               1.41                                                  A--C--N--S--F--NH.sub.2                                               AP17    R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               7.35                                                  A--Q--C--N--S--F--R--Y                                                AP20    R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               7.50                                                  A--C--N--S--F--R--Y                                                   AP2l    R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               15.15                                                 C--N--S--F--R--Y                                                      AP22    R--S--S--C-- F--G--G--R--I--D--R--I--                                                                 >40                                                   C--N--S--F--R--Y                                                      __________________________________________________________________________

In the compounds of both Tables 1A and 1B, X₁ is R-S-S. Table 1C showsthat the presence of R-S-S or, indeed, the presence of X₁ per se, isunnecessary.

                                      TABLE 1C                                    __________________________________________________________________________    Peptide Sequence                Ki(app)(nM)                                   __________________________________________________________________________    rANP(126-150)                                                                         R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               7.26                                                  A--Q--S--G--L--G--C--N--S--F--R--Y                                    AP20    R--S--S--C--F--G--G--R--I--D--R--I--G--                                                               7.50                                                  A--C--N--S--F--R--Y                                                   AP36    C--F--G--G--R--I--D--R--I--G--A--C                                                                    14.92                                         AP37    C--F--G--G--R--I--D--R--I--G--A--C--NH.sub.2                                                          13.40                                         AP62    C--F--G--G--R--I--D--R--I--G--A--C--                                                                  5.96                                                  N--S--F--NH.sub.2                                                      AP109  R--C--F--G--G--R--I--D--R--I--G--A--C--                                                               4.97                                                  N--S--F--NH.sub.2                                                      AP110  C--F--G--G--R--I--D--R--I--G--C--N--S--                                                               6.51                                                  F--NH.sub.2                                                           AP64    C--R--I--D--R--I--G--A--Q--S--G--L--G--                                                               12.39                                                 C--NH.sub.2                                                           AP67    C--F--G--G--R--I--D--R--I--G--A--Q--S--                                                               7.08                                                  G--C--NH.sub.2                                                        AP69    C--F--G--G--R--I--D--R--I--G--A--Q--                                                                  2.45                                                  C--NH.sub.2                                                           AP65    C--R--I--D--R--I--G--A--Q--S--                                                                        14.04                                                 G--L--C--NH.sub.2                                                     AP70    Y--C--F--G--G--R--I--D--R--I--G--A--                                                                  7.07                                                  C--NH.sub.2                                                           AP83    C--F--G--G--R--I--D--R--I--G--A--                                                                     5.75                                                  C--NH.sub.2                                                           AP91    C--F --G--G--R--I--D--R--I--G--                                                                       24.03                                                 A--C--NH.sub.2                                                        AP84    C--F--A --G--R--I--D--R--I--G--                                                                       13.29                                                 A--C--NH.sub.2                                                        AP85    C--F--S --G--R--I--D--R--I--G--A--                                                                    5.25                                                  C--NH.sub.2                                                           AP86    C--F--L --G--R--I--D--R--I--G--                                                                       14.04                                                 A--C--NH.sub.2                                                         AP111  C--F--V G--R--I--D--R--I--G--A--                                                                      10.68                                                 C--NH.sub.2                                                            AP112  C--F--Aib--G--R--I--D--R--I--G--A--                                                                   69.57                                                 C--NH.sub.2                                                           AP82    C--F--G--G--R--I--D-- R--I--G--Aib--                                                                  4.43                                                  C--NH.sub.2                                                            AP113  C--F--G--G--R--M --D--R--I--G--                                                                       15.77                                                 A--C--NH.sub.2                                                        AP94    C--F--G--G--R--I--D--R--I--A --                                                                       26.73                                                 A--C--NH.sub.2                                                        AP95    C--F--G--G--R--I--D--R--I--G--A--                                                                     20.34                                                 A--C--NH.sub.2                                                        AP96    C--F--G--G--R--I--D--R--I--G--A--                                                                     3.75                                                  C --NH.sub.2                                                          AP89    C--F--G--G--R--I --D--R--I--G--                                                                       >100                                                  A--C--NH.sub.2                                                        AP90    C--F--G--G--R--I--D --R--I--G--                                                                       >100                                                  A--C--NH.sub.2                                                        AP92    C--F--G--G--R--I--D--R --I--G--                                                                       >100                                                  A--C--NH.sub.2                                                         AP114  C--A--G--G--R--I--D--R--I--G--A--                                                                     >160                                                  C--NH.sub.2                                                           __________________________________________________________________________

Table IC shows data for additional compounds, including one compound(AP96) wherein one of the C residues is in the D form. Many of thesecompounds exhibit significant receptor binding activity. However, thediminished binding affinity of less preferred embodiment AP114 iscurrently under investigation. Alternations in the native sequence seemless well tolerated as ring size is reduced and extra-ring extensionsare removed. In addition, in some instances wherein the R(I/M)DRIpentapeptide core is altered, affinity is also greatly diminished.

Limitations on alterations of this core are shown in the series AP113,AP89, AP90 and AP92. Substitution of the D optical isomer for the Lamino acid appears to diminish activity in all instances except in thecase of M as AA9 and AP113. Attention is called, in particular, to thedemonstration, for example, for AP36, that both X₁ and X₄ may be absent,and for AP112 and AP36, for example, X₂ and X₃ together may represent asfew as 5 amino acids.

To some extent, however, reduction in ring size and/or inclusion ofcysteine in the D form is mitigated by inclusion of a hydrophobicresidue within the required distance, as shown in Table 1D.

                  TABLE 1D                                                        ______________________________________                                                                       Ki(app)                                        Peptide                                                                             Sequence                 (nM)                                           ______________________________________                                        AP577 (2-NA)-C-G-R-I-D-R-I-G-A-C-NH.sub.2                                                                    30.0                                           AP578 (2-NA)-G-C-R-I-D-R-I-G-A-C-NH.sub.2                                                                    24.0                                           G-R-I-D-R-I-G-A-C-NH.sub.2                                                          35.3                                                                    R-I-D-R-I-G-A-C-NH.sub.2                                                            110.5                                                                   NH.sub.2-NA)-G-C-R-I-D-R-I-G-A-C                                                    21.2                                                                    NH.sub.2-NA)-C-G-R-I-D-R-I-G-A-C                                                    21.9                                                                    NH.sub.2R-I-G-A-C                                                                   109.1                                                                   NH.sub.2R-I-G-C                                                                     104.1                                                                   NH.sub.2I-G-C -C                                                                    140.0                                                                   NH.sub.2I-G-A-C                                                                     124.0                                                                   G-R-I-D-R-I-G-C-NH.sub.2                                                            39.5                                                                    AP588 (2-NA)-C-G-R-I-D-R-I-G-C-NH.sub.2                                                                      6.0                                            AP589 (2-NA)-G-C-R-I-D-R-I-G-C-NH.sub.2                                                                      73.5                                           AP540 (2-NA)-C-G-R-I-D-R-I-C-NH.sub.2                                                                        >400                                           AP541 (2-NA)-G-C-R-I-D-R-I-C-NH.sub.2                                                                        393.0                                          R-I-D-R-I-G-C-NH.sub.2                                                              375.0                                                                   ______________________________________                                    

The compound AP543 demonstrates that the bridge need not be a disulfide:##STR6##

This compound without a disulfide ring but wherein X₁ is hydrophobic ishighly active.

In order to show that the receptor binding assay is specific to ANP,data which compares ANP-receptor interactions of rANP(126-150) with theuunrelated peptide hormones angiotensin II, glucagon, parathyroidhormone and gamma-MSH is shown:

    ______________________________________                                        Peptide           Ki(app)                                                     ______________________________________                                        rANP(126-150)     7.50                                                        angiotensin II    >500                                                        glucagon          >500                                                        parathyroid hormone                                                                             >500                                                        gamma-MSH         >500                                                        ______________________________________                                    

As shown above, only rANP(126-150) displays detectable ANP-receptoraffinity. This attests to the relevant ANP-specificity of this receptor.

The data in the foregoing tables show that a large representative sampleof the compounds of the invention demonstrate affinity in the sspecificreceptor-binding assay described. The following subsections demonstratethat representative compounds, which demonstrate receptor binding, arealso active in vivo, whereas compounds which do not bind to thesereceptors appear inactive.

B. WHOLE MAMMAL BIOASSASY

The biological activity of analog compounds of the present invention canbe demonstrated in anesthetized rats and dogs. The correlation ofreceptor binding affinity annd in vivo effects demonstrates thepredictive value of the receptor assays for biological activity.

1. DIURESIS IN ANESTHETIZED RATS

In one set of examples, cannulae were placed in the left and rightureters and femoral vein of anesthetized rats and urine was collectedfrom the ureters. Analog peptides were administered via the femoralvein. Prior to infusing the analog peptides, saline was infused for 30minutes, urine was collected for 6 five-minute baseline periods andurine volume was determined gravimetrically.

Following these baseline collection periods, various analog peptideswere infused for 30 or 60 minutes and urine volume was measured infive-minute periods during infusion and for 60 minutes followinginfusion (at which time rats were returned to saline). Data was examinedby averaging urine flow rates for six five-minute baseline controlperiods immediately preceding infusion, and comparing values during andafter administration of peptides with the "baseline" control values.Responses to peptides are thus evaluated and plotted as the percent ofbaseline control responses. Specific examples are shown in FIGS. 3A-H.The error bars at the beginning of the graphs represent baseline values± standard deviations. Responses to peptides that are substantiallyabove baseline±SD can thus be interpreted as being statisticallysignificant increases.

As shown in FIGS. 3A-H, diuretic responsive correlate with predictionsfrom receptor-binding studies. Analog peptides AP25, AP20, AP21, AP37,AP101, AP319 and AP324, significantly increased urine flow rate (urinevolume) when infused at 5 mcg/min, 5 mcg/min, 5 mcg/min, 10 mcg/min, 5mcg/min, 10 mcg/min and 10 mcg/min respectively. Their respective Kivalues are respectively 8.62, 7.50, 15.15, 13.40, 2.63, and 6.0.

On the other hand, AP54, which falls outside the scope of the compoundsof the invention, and which was shown in Table 1A to lack significantreceptor-binding activity, also appears inactive in vivo. These data,therefore, demonstrate the appropriate correlation betweenreceptor-binding activity and in vivo activity.

2. DIURESIS AND NATRIURESIS IN ANESTHETIZED DOGS

The biological activity of analog compounds of the present invention canalso be demonstrated in pentobarbital-anesthetized dogs. In theseexamples, cannulae were placed in the left and right ureters and femoralvein of anesthetized dogs and urine was collected from the ureters.Analog peptides were administered via the femoral vein. Prior toinfusing analog peptides, saline was infused for 30 minutes and urinewas then collected for three ten-minute collection periods. Urine volumewas determined gravimetrically and urine sodium was determinedphotometrically.

Following these three baseline collection periods, the selected analogpeptides were infused for 60 minutes and urinary flow was measured foran additional 60 minutes following infusion. During infusion (60minutes) and recovery (60 minutes), ten-minute collection periods wereobtained. Control animals which received only saline were studied inparallel.

Data were examined by comparing urine flow rates for dogs infused withpeptides AP101 (3 mcg/kg/min), AP306 (3 mcg/kg/min), AP324 (3mcg/kg/min), or AP314 (1 mcg/kg/min) against control animals infusedwith saline.

                  TABLE 2A                                                        ______________________________________                                               Dose     Peak Urine  Peak Urine                                               (mcg/kg/ Flow Rate   Sodium Excretion                                  Peptide                                                                              min)     V (ml/min)  U.sub.Na V (mcEq/min)                                                                     Ki                                    ______________________________________                                        Control                                                                              --       0.61        40                                                AP101  3        1.77*                   2.63                                  AP306  3        2.09*                   2.03                                  AP314  1        2.05*       365*        14.5                                  AP324  3        2.47*       495*        48.5                                  ______________________________________                                    

As shown in Table 2A, the peak in vivo responses to the peptides aresubstantially above baseline (*P<0.05 by Student's test) and areinterpreted as being statistically significant increases, comparable tothe effects of native ANP compounds.

Diuretic and natriuretic responses to peptides AP101, AP306, AP314 andAP324 correlate with predictions from receptor-binding assays. Analogpeptides AP101, AP306, AP314 and AP324, each of which is a small linearpeptide with substantial Ki(app)s (Table 1B), significantly increasedurine flow rate (urine volume) and urinary sodium excretion when infusedat 3, 3, 1 or 3 mcg/kg/minute, respectively. Thus, these analog peptidesare shown to each induce diuresis and natriuresis and therefore supportthe predictive value of the receptor-binding analysis for diureticpotency.

3. BLOOD PRESSURE RESPONSES IN ANESTHETIZED RATS

Compounds of the present invention also lowered blood pressure whenadministered as a bolus or infusion to anesthetized rats. Table 3Bpresents data which compares the blood pressure effects ofrepresentative analog peptides AP20 and AP37, with that ofrANP(126-150), following administration by infusion.

                  TABLE 2B                                                        ______________________________________                                                                Dose        Blood                                     Peptide                                                                              Structure        (p mol/kg/min)                                                                            Pressure                                  ______________________________________                                        rANP   R-S-S-C-F-G-G-   183         -39 ± 5                                (126-150)                                                                            R-I-D-R-I-G-A-Q-                                                              S-G-L-G-C-N-S-F-R-Y                                                    AP20   R-S-S-C-F-G-G-   733         -34 ± 3                                       R-I-D-R-I-G-A-                                                                C-N-S-F-R-Y                                                            AP37   C-F-G-G-R-I-D-   7330        -14 ± 3                                       R-I-G-A-C-NH.sub.2                                                     ______________________________________                                    

As shown, each of the analog peptides lowered blood pressuresignificantly. While analog peptide AP37 exhibited a weaker effect onblood pressure at a substantially higher dose (40x that ofrANP(126-150)), it nevertheless was hypotensive. It has also been foundthat analog peptides AP40, AP41 and AP57 exhibit activity similar toAP37.

Again, compounds which showed receptor-binding affinity are shown to beactive in vivo. The correlation between the in vitro receptor-bindingtest and in vivo results further supports the validity of thereceptor-binding assay to show the therapeutic efficacy of the compoundstested.

Thus, the administration to mammalian hosts of therapeutically effectiveamounts of the additional disclosed analog peptides, or pharmaceuticalcompositions containing these analog peptides, can be used tosubstantially increase natriuresis and diuresis and/or alter thevascular caliber. Furthermore, administration of selected analogpeptides within the scope of the present invention can be used to treatcases of hypertension or various edematous states whose etiology doesnot require the full range of biological activity provided by native ANPcompounds.

C. ISOLATED TISSUE BIOASSAYS

It is believed, as stated above, that the effect of the analogs of theinvention herein in vivo is due to their ability to potentiate theeffect of endogenous ANP, possibly through blockage of the receptorsinvolved in binding and clearing endogenous ANPs. Accordingly, one wouldexpect that the diuretic and natriuretic effects of the analogs would bediminished or eliminated in isolated tissue where ANPs are not presentunless specifically supplied. The results below demonstrate support forthis theoretical model. As shown below, while a representative analog,AP57, was active in vivo, it was not active in isolated perfused ratkidney. However, in the same model system, it was able to potentiate theeffect of rANP(123-150).

1. NATRIURESIS AND DIURESIS IN THE ISOLATED PERFUSED RAT KIDNEY

The biological actions of the ANP analogs can be demonstrated in theisolated perfused rat kidney, as described in Camargo, M. J. F., et al,Am J Physiol (1984) 246: F447-F456. In a particular set of examples, theeffect of the 15-amino-acid peptide R-S-S-C-F-G-G-R-I-D-R-I-G-A-C-NH₂(peptide AP57) at a concentration of 10⁻⁷ M, was demonstrated in theintact kidney. The results are in Table 2C.

                                      TABLE 2C                                    __________________________________________________________________________    EFFECT OF AP57 ON DOSE-RESPONSE CURVES OF                                     rANP(123-150) IN THE ISOLATED PERFUSED RAT KIDNEY                             GFR         FF    RVR      U.sub.NA V                                                                            FE.sub.Na V                                (ml/min)    (%)   (mmHg/ml/min)                                                                          (uEq/min)                                                                             (% ul/min)                                 __________________________________________________________________________    A. CONTROL KIDNEYS (N = 5)                                                    0.48 ± 0.06                                                                      1.21 ± 0.15                                                                      2.30 ± 0.12                                                                      0.37 ± 0.08                                                                         0.56 ± 0.0916.5 ± 2.0                        B. AP57 (10.sup.-7 M) (N = 4)                                                 0.55 ± 0.07                                                                      1.37 ± 0.22                                                                      2.27 ± 0.17                                                                      0.17 ± 0.04                                                                         0.24 ± 0.0813.8 ± 2.0                        C. rANP(123-150) (10.sup.-11 TO 10.sup.-8 M) (N = 4)                          [C]   0.45 ± 0.06                                                                      1.22 ± 0.20                                                                      2.29 ± 0.15                                                                         0.30 ± 0.050.50 ± 0.1015.0 ± 1.8          10.sup.-11                                                                          0.53 ± 0.04                                                                      1.33 ± 0.20                                                                      2.36 ± 0.20                                                                         0.53 ± 0.140.72 ± 0.1921.8 ± 1.7          10.sup.-10                                                                          0.66 ± 0.07                                                                      1.76 ± 0.29                                                                      2.51 ± 0.26                                                                         1.13 ± 0.431.21 ± 0.4435.5 ± 5.7          10.sup.-9                                                                           0.82 ± 0.07                                                                      2.30 ± 0.41                                                                      2.64 ± 0.35                                                                         3.22 ± 0.922.78 ± 0.8974.0 ± 14.8         10.sup.-8                                                                           0.80 ± 0.08                                                                      2.23 ± 0.44                                                                      2.66 ± 0.32                                                                         4.78 ± 1.154.12 ± 0.9496.6 ± 24.0         D. rANP(123-150) (10.sup.-11 TO 10.sup.-8 M) IN PRESENCE OF AP5 &             (10.sup.-7 M).sup.3 (N = 4)                                                   [C]   0.65 ± 0.06                                                                      1.57 ± 0.27                                                                      2.21 ± 0.21                                                                         0.17 ± 0.050.18 ± 0.0416.3 ± 2.0          10.sup.-11                                                                          0.80 ± 0.08                                                                      2.04 ± 0.15                                                                      2.50 ± 0.15                                                                         0.45 ± 0.100.41 ± 0.1131.0 ± 3.7          10.sup.-10                                                                          0.93 ± 0.04                                                                      2.65 ± 0.20                                                                      2.80 ± 0.31                                                                         1.76 ± 0.251.33 ± 0.2070.6 ± 4.2          10.sup.-9                                                                           0.99 ± 0.07                                                                      2.81 ± 0.40                                                                      2.79 ± 0.34                                                                         6.03 ± 1.374.08 ±  0.72139 ± 14           10.sup.-8                                                                           0.84 ± 0.13                                                                      2.30 ± 0.47                                                                      2.63 ± 0.33                                                                         6.47 ± 2.294.77 ± 1.22132                    __________________________________________________________________________                               ± 33                                             GFR = glomerular filtration rate; FF = filtration fraction; RVR = renal       vascular resistance; U.sub.Na V = urinary sodium excretion rate; FE.sub.N     = fractional sodium excretion: V = urine flow rate. Results are presented     as mean ± SE, with the number of kidneys presented for each test phase     In the phase demonstrating the effect of AP57 on the dose response curve      of rANP(123-150), 10.sup.-7 M AP57 was added 30 minutes before the            addition of increasing doses of rANP(123-150).                           

Despite having natriuretic and diuretic activities in an intact rat,analog peptide AP57 was not active in the isolated perfused rat kidneyat a concentration of 10⁻⁷ M, as compared to rANP(123-150), as shown insections A and B of the table.

Sections C and D of the table show the ability of peptide AP57 tomodulate the renal responses to the rANP(123-150). As shown in Table 2C,section C, rANP(123-150) increases glomerular filtration rate, renalvascular resistance, filtration fraction, urinary sodium excretion rate,fractional excretion of sodium and urinary flow rate in a dose-dependentmanner in the concentration range from 10⁻¹¹ to 10⁻⁷ M. Table 2C,section D, shows that pretreatment of the isolated kidney with 10⁻⁷ MAP57 causes the subsequent responses to rANP(123-150) to occur atreduced concentrations. Analog peptide AP57, despite being apparentlyinactive in this in vitro model, increased the potency of rANP(123-150).Subsequent assays have confirmed these observations and conclusions.

Table 2D shows that both rANP(123-150) and AP57 compete for specific(¹²⁵ I)-rANP(123-150) binding to the cortex of the kidney.

                  TABLE 2D                                                        ______________________________________                                        RATIO OF BOUND/FREE (.sup.125 I)-rANP(123-150)                                           WHOLE KIDNEY                                                                              OUTER CORTEX                                           ______________________________________                                        (.sup.125 I)-rANP(123-150)                                                                 122 ± 46   176 ± 23                                        (4 × 10.sup.-12 M)                                                      (n = 3)                                                                       (.sup.125 I)-rANP(123-150)                                                    (4 × 10.sup.-12 M)                                                        +          0.63 ± 0.27                                                                              0.77 ± 0.30                                     rANP(123-150)                                                                 (10.sup.-7 M)                                                                 (n = 3)                                                                       (.sup.125 I)-rANP(123-150)                                                    (4 × 10.sup.-12 M)                                                        +          1.27 ± 0.32                                                                              1.68 ± 0.44                                     AP57                                                                          (10.sup.-7 M)                                                                 (n = 3)                                                                       ______________________________________                                    

These cortical-associated receptor sites may be involved in theclearance and removal of the ANPs. Thus, AP57 may block the clearance ofrANP(123-150) in the isolated kidney model, thereby explaining itsability to potentiate the effects of rANP(123-150). Furthermore, if AP57blocks clearance of endogenous ANPs, it may explain the natriuretic,diuretic and vasorelaxant responses to these peptides in vivo.

Although the foregoing invention has been illustrated above for purposesof aiding understanding, modifications of the invention may be practicedwhile remaining within the spirit and scope of the appended claims.

We claim:
 1. A peptide compound having natriuretic, diuretic and/orvasodilator activity in mammals, which has the formula:

    X.sub.1 A.sub.y X.sub.2 -AA.sub.8 -AA.sub.9 -AA.sub.10 -AA.sub.11 -AA.sub.12 -X.sub.3 A.sub.z X.sub.4                       ( 1)

wherein: each of AA₈ and AA₁₁ is, independently, a basic/noncyclic;neutral/nonpolar/small; or neutral/polar/large/nonaromatic amino acidresidue; AA₉ is a neutral/nonpolar/large/nonaromatic amino acid residuein the D or L configuration; AA₁₀ is an acidic amino acid residue; AA₁₂is a neutral/nonpolar/large/nonaromatic amino acid residue in the D or Lconfiguration or a covalent bond; AA_(y) and AA_(z) are amino acidresidues which together form a bridging bond, said bond selected fromthe group consisting of a disulfide bond, a methylene bond, asulfide/methylene bond, an amide bond and an ester bond; X₁ is H, acetyla peptide of from 1 to 125 amino acid residues, or the desNH₂ formthereof or is a hydrophobic aliphatic, aromatic, or mixedaliphatic/aromatic organic group of from 6 to 20 carbon atoms; X₂ is abond or peptide or 1-10 residues, provided the distance between theamino group of AA₈ and a hydrophobic moiety occuring in X₁ AA_(y) X₂ isbetween about 4.5 and 15 A in an achievable conformation; X₃ is a bondor a peptide of 1-10 residues, and X₄ is (OH), NH₂, NHR' or NHR'R"wherein R' and R' are straight or branched chain alkyl (1-10C) wherein1-2 nonadjacent C may be replaced by N, O, or S, or X₄ is a peptide offrom 1 to 20 residues, including the C-terminal amide or alkyl amideforms thereof or is absent; wherein the total ring size is equivalent tothat obtained by disulfide bridge formation between cysteine residuesseparated by 5-15 alpha-amino acids; wherein one or more of the amidelinkages between adjacent amino acid residues may optionally be replacedby a linkage selected from the group consisting of --CH₂ NH--, --CH₂--S--, --CH₂ CH₂ --, --CH═CH--, --COCH₂ --, --CH(OH)CH₂ -- and --CH₂SO--, and with the proviso that if A₁₂ is not a bond, and X₂ is atripeptide, X₃ cannot be a heptapeptide.
 2. The compound of claim 1wherein X₁ is a hydrophobic aliphatic, aromatic or mixedaliphatic/aromatic organic group of from 6 to 20 carbon atoms.
 3. Thecompound of claim 1 wherein X₁ is selected from the group consisting offluorenylmethyl oxycarbonyl, benzyloxycarbonyl, 2-(2'-(6'-methoxynaphthyl)) propionyl, diphenylpropionyl, biphenylacetyl,triphenylpropionyl, cyclohexylacetyl, 3-indolepropionyl,4-indolebutyryl, 1-adamantylacetyl, 1-naphthylacetyl, 2-naphthylacetyl,1-naphthoxyacetyl, 2-naphthoxyacetyl, dibenzylacetyl,bis(1'-naphthylmethyl)acetyl, 2-naphthyl thioacetyl, 3-phenoxypropionyl,2-naphthoyl, 2-naphthoxy, 2-naphthyl, phenylalanyl and des-NH₂-phenylalanyl.
 4. The compound of claim 3 wherein X₁ is selected fromthe group consisting of 2-naphthylacetyl, 2-naphthoxyacetyl,1-naphthylacetyl, phenylalanyl and des-NH₂ phenylalanyl.
 5. The compoundof claim 1 wherein X₁ is hydrogen or a peptide of from 1 to 6 amino acidresidues, said peptide selected from the group consistingof:S-L-R-R-S-S, L-R-R-S-S, R-R-S-S, R-S-S, S-S, and S, and the foregoingwherein one residue is replaced by another residue of its same subclass.6. The compound of claim 1 wherein X₂ is selected from the groupconsisting of a bond, and a peptide of 1-2 amino acids which are of thesubclasses neutral/polar/small or neutral/nonpolar/small.
 7. Thecompound of claim 5 wherein X₂ is selected from a bond or a peptide of1-3 amino acids which are of the subclasses neutral/polar/small orneutral/nonpolar/small.
 8. The compound of claim 5 wherein X₂ isF-(AA)_(a) wherein a is 1 or 2 and AA is of the subclassesneutral/polar/small or neutral/nonpolar/small.
 9. The compound of claim1 wherein X₃ is selected from the group consisting of:G-A-Q-S-G-L-G,G-A-Q-S-G-L, A-Q-S-G-L-G, G-A-Q-S-G, Q-S-G-L-G, G-A-Q-S, S-G-L-G, G-A-Q,G-A-A, G-L-G, L-G, G-A, G and a bond, and the foregoing wherein oneresidue is replaced by another residue of the same subclass; and--NH(CH₂)_(b) CO-- wherein b is 3-6.
 10. The compound of claim 1 whereinX₄ is (OH) NH₂, NHR' wherein R' is straight or branched chain allyl of1-10C, wherein 1-2 nonadjacent C can be replaced by N, O, or S, or is apeptide of 1-5 amino acid residues or the amide or alkylamide thereof.11. The compound of claim 10 wherein X₄ is selected from the groupconsisting of:N-S-F-R-Y, N-S-F-R, N-S-F, N-S, N or OH and the amides oralkylamides thereof, and the foregoing wherein one residue is replacedby another residue of the same subclass.
 12. The compound of claim 1wherein at least one of the amide linkages between adjacent amino acidresidues is replaced by a linkage selected from the group consisting of--CH₂ NH--, --CH₂ --S--, --CH₂ CH₂ --, --CH═CH-- (cis and trans),--COCH₂ --, --CH(OH)CH₂ -- and --CH₂ SO--.
 13. The compound of claim 1wherein AA₈ -AA₉ -AA₁₀ -AA₁₁ -AA₁₂ is R(I/M)DRI or at most one residuetherein is replaced by substitutingA, Q, N, K, L or NMeIle for R as AA₈V, V , L, L , I , M , t-BuA, t-BuG or Cha for I or M as AA₉ ; E or Cya,for D as A₁₀ ; A, Q, N, K, Orn or Cit for R as A₁₁ ; and M, M , V, V ,L, L , I , N-MeIle, t-BuA or a covalent bond for I as AA₁₂.
 14. Thecompound of claim 1 wherein AA₈ -AA₉ -AA₁₀ -AA₁₁ -AA₁₂ is selected fromthe group consisting of:

    ______________________________________                                        A(I/M)DRI RM DRI      R(I/M)DRL                                               K(I/M)DRI RLDRI       R(I/M)DRM                                               Q(I/M)DRI R(I/M)ERI   R(I/M)DRM                                               RVDRI     R(I/M)DKI   R(I/M)DRI                                               RI DRI    R(I/M)DQI   R(I/M)DRV and R(I/M)DRI.                                ______________________________________                                    


15. The compound of claim 1 which is selected from the group consistingof that shown in FIG.
 4. 16. A composition useful as a natriuretic,diuretic and/or vasodilator comprising a therapeutically effectiveamount of the compound of claim 1 together with a pharmaceuticallyacceptable carrier.
 17. A method for inducing natriuresis, diuresis, orvasodilatation in a mammalian host, which comprises administering tosaid host a pharmaceutically effective amount of the composition ofclaim
 16. 18. A process for production of a peptide compound havingnatriuretic, diuretic and/or vasodilator activity in mammals, saidpeptide compound having the formula of the compound of claim 1, or thepharmacologically acceptable salts thereof, which process comprises thefollowing steps:a. preparing a protected peptide bonded to a solid resincarrier in a reaction mixture, wherein the peptide has an amino acidsequence as recited above; b. removing the solid resin carrier from thepeptide and deprotecting the peptide; c. optionally forming a ringcompound from the peptide; d. optionally modifying the peptide to addany desired organic substituent groups as recited above; and e.isolating the peptide from any reaction mixture, and optionally,converting the polypeptide into an acid addition salt thereof.
 19. Thecompound of claim 1 wherein X₁ is H, acetyl, or a peptide of 1-6 aminoacid residues or the desNH₂ form thereof or is a hydrophobic aliphatic,aromatic, or mixed aliphatic/aromatic organic group of from 6-20 carbonatoms.
 20. The compound of claim 19 wherein said peptide is selectedfrom the group consisting of R, Y, S, SS, RSS, YSS, RRSS, LRRSS, andSLRRSS.